Pregnancy antigen



Patented Mar. 28, 1939 I UNITED STATES PATENT OFFICE PREGNANCY ANTIGENBenjamin Gruskin, Philadelphia, Pa., assignor to Lakeland Foundation,Chicago, Ill., a corporation of Illinois No Drawing. Application April29, 1936, Serial N0. 76,874

10 Claims. (01.167-48.)

This invention relates to a substance commonly tion sometimes occursduring pregnancy and it. is referred to as antigen, and relates to anantigen at such times that a diagnosis disclosing the fact for thedetermination of pregnancy. of pregnancy is of great importance. Whenusing This application relates to certain improvean antigen extractedfrom both the maternal ments over applicants co-pending application, andthe foetal layers of the placenta, in accord- Serial No. 731,117, filedJune 18, 1934, which has ance with one specific aspect of my inventiondismatured into United States Patent No. 2,042,430, closed and claimedbroadly in my U. S. patent patented May 26, 1936. No. 2,042,430, thetest is contra-indicated during The present invention relates morespecifically menstruation, because of the decidual involve- 0 to anantigen and test for determining pregnancy ment of that process whichwill respond to the wherein the antigen is made solely from thehomologous protein of the placenta extract givfoetal layer of theplacenta whereby a strong ing positive reactions. I have now discoveredpseudopodic skin reaction occurs when the that the antigen produced fromthe foetal layer antigen is injected intradermally in a pregnant only isfree from any homologous protein which mammalian, the purpose beingprimarily to prowill cause a false reaction at this time. 15

vide an antigen which gives a strong and sharp The antigen reaction sothatearly and late pregnancy can be easily and e t l t i Placentas areobtained from knownhealthy An important object of the present inventionW m -n v h m n ei patients I is to provide an antigen and-test which iscapable the test i to be performed 011 animals, the 20 of accuratelydetermining th fact of pregnancy placentas used are'obtained from acharacteristic at all times and even during menstruation. mammalianplecentas t be P p Another object of the invention is to provide Wi n hur f r liv y, nl hey an be an antigen for the purpose of detectingwhether frozen until used. The object of this is to prevent or notpregnancy exists by the injection intraautolysis occurring in the cellsof the placenta. 25 dermally of an antigen which is made from the r Theplacenta is washed free of blood, the cord foetal layer of the placenta,particularly from the Cut and the emnienie See 0111, y. and placentafrom t m t giving bi t t 11 all loose tissue removed from the surface ofthe male. placenta. The placenta is then placed in a dish 0 Numerousother objects and advantages will With Curved inner Surface, Such as alarge be apparent throughout the progress of the folevaporating dish, Pa t oe de Of the lowing speciflgatiom placenta next to the dish. Theplacenta is then The theory upon which the present invention frozen, SayOvernight, in e dish- After the and discovery is based, consists in thefact that tissue is thoroughly fr n. h pl en a is the foetal layer ofthe. placenta contains a charv d from the dish- The layer of amnionic 35acteristic protein specific to the embryonic foetal tissue adhering tothe foetal side of the placenta protein in the system of a, pregnantmammalian, iS carefully dissected Ofi, and when this has been and t twhen an antigen is made up of an removed, with a sharp scalpel thefoetal layer of tract of the foetal layer of the placenta and introtheplacenta is Ofi 110 a dep f a ut n 40 duced intradermally into apregnant mammalian half centimeter- The large blood vessels should 40the characteristic proteiii will respond allergically not be includedwith the tissue to be used- After and produce pseudopodia, Therefore anantigen the foetal tissue has been cut oil, it should be made up offoetal placenta introduced into the Washed free of blood This s most sly d skin of a pregnant female will cause a p'seudoby Placing the tissueinlay Piece Of bolting 610th,

podic formation to occur, this formation appearand a n the s u n ruWeller- After 45 ing about the margin of the wheal formed by the alltraces of blood have been washed from the injection. When using theantigen made up by tissue, the tissue is placed in three volumes ofextracting tissue comprising the foetal layerof a acetone, Which isPoured Ofi after tW0 hours, a mammalian'placenta, which tissue is freeof any fresh quantity of acetone added, and he tissue part of thematernal layer, it has been found that allowed to remain in the acetonefor twenty-four 50 new and valuable results are obtained. Thus, anhours, after which time the acetone is discarded, antigen prepared inaccordance with the present and the tissue thoroughly dried. inventionwill accurately determine the fact of The placenta as a whole may beused for pregnancy during menstruation. It is known, in antigen asdisclosed in my aforesaid prior patent,

accordancewith medical science, that menstruaor I may use the part ofthe placenta remaining 55 after removing the amnionic tissueand removingthe maternal layer to a depth of one-half centimeter. by grinding thetissue in a cone type grinder, washing it free of blood, and drying thetissue in acetone. However, it has been found that use of the foetallayer alone will eliminate the possibility of false positive reactionsduring menstruation in cases of endometritis, etc.

Very excellent results have been obtained by the use of the foetal layerof the placenta, and it has been found that other excellent and ve ysharp results are obtained in accordance with thepresent invention whenthe foetal layer of the placenta used is that obtained from the placentaof a born female.

The dried tissue from the foetal layer is then ground to a coarsepowder, and is extracted with a one-tenth normal solution ofsodium-hydroxide, in the proportion of 1.5 grams of dry tissue to 100cc. of tenth normal sodium-hydroxide. The tissue is mixed thoroughlywith the sodiumhydroxide in a mortar and rubbed up, and is then placedin a glass stoppered Pyrex cylinder. The solution is shaken severaltimes during the day, so that the tissue will be thoroughly extracted,as the tissue has a tendency to settle to the bottom of the tube, andunless it is thoroughly shaken with the solution, complete extractionwill not take place. The tissue and sodiumhydroxide solution are allowedto stand for twenty-four hours in the cylinder. After this time theextract is poured into Pyrex tubes and centrifuged for five minutes atspeed nine (on an International centrifuge). The supernatant solution isthen pipetted off into a Pyrex beaker and the remaining tissue isdiscarded. The solution is then neutralized to a pH of 6.9 with asolution of 0.05 normal HCl containing 2.27 grams of anhydrous KHzPOtper liter. The solution used for neutralizing and comprising 0.05 normal1101 containing 2.27 grams of anhydrous KHzPOr per liter is termed anacid and buffer solution.

The acid and buffer solution just described is added to the alkalineextract which has been pipetted off from the cells and measured. The

acid and buffer solution should be added slowly and the solutionscarefully stirred or gently agitated while the acid and buffer solutionis being added. After the acid and buffer solution has been added in anamount equal to the alkaline extract of the tissue, a few more cubiccentimeters of the acid and buffer solution maybe required. Theresultant solution should then betested to see if the neutralization isnearing the end point. This testing should be repeated frequently tomake sure that the nitration does not go past the end point, which is ofa pH of 6.9 for this process. If the resultant solution is made tooacid, the protein of the antigen will be precipitated. The antigenshould be checked electrometrically or it may be checked against astandard solution of a pH of 6.9, using the spot plate method, withbrom-thymol-blue as an indicator.

As a comparison standard for the pH determination when the spotplatemethod is used with brom-thymol-blue as the indicator, a solu tion ofanhydrous KH2PO4 and NazHPO4 is employed in the following proportions:

The amounts mixed for the pH standard are of producing pseudopods innormal cases.

4.9 cc. or KHaPOl and 5.1 cc. of NazI-IPO; solution.

The antigens and control solutions must be kept in Pyrex containers, asother glass gives up alkalis to the solutions which changes the pH andmakes the antigen unusable.

The neutralized antigen is placed in sterile Pyrex containers, withrubber stoppers which have been boiled for fifteen minutes to removesoluble impurities. A preservative of one part tricresol to two partsglycerin is then added to the antigen, two drops of the mixture to eachfive cc. of antigen, which is then thoroughly shaken, so that thepreservative will be thoroughly in solution. The stoppers of the vialsshould be pierced with a r cdle, to permit the compressed air to escapefrom the vials. The antigen is then placed in the ice chest fortwentyfour hours, after which a slight sediment is sometimes noticed. Ifthere should be a slight sediment present, pipette off the antigen intoa sterile vial, and recheck the pH of the solution. The antigen willhave a slight colloidal opacity, but must be free from anyprecipitation.

Test

Qne-tenth of one cubic centimeter of the above antigen is drawn ofi intoa small syringe to which there is attached a very fine short needle. Theantigen is injected intradermally, after first sterilizing and treatingthe surface of the patients skin and rendering it perfectly dry. Theinjection is performed by stretching the skin with one hand andinjecting the antigen intradermally, the injection being made by theusual intradermal method. In positive cases, that is, in cases where thepatient examined is pregnant, a slight area of reaction will be noticedsurrounding the small bubble, termed a wheal, which wheal occurs fromthe injection, and pseudopods will form. Pseudopods are radialelongations extending outwardly from the edges of the wheal. In negativecases, that is, in cases where the patient is not pregnant, no pseudopodformation will take place.

The above test is based on the fact that since the placenta is made upof the foetal layer, a protein characteristic of foetal embryoniccharacter is partly absorbed into the system of the mother. When anextract is made up of the foetal layer of the placenta and this extractis injected intradermally, pseudopods will be formed because thespecific foetal embryonic character of the antigen is homologous to thespecific embryonic foetal protein of the mother, according to the lawsof protein sensitization or allergy. The antigen is furthermore freefrom all proteins which would be homologous 'to any decidual involvementof the process of menstruation and, for this reason, the formation ofpseudopods shall occur only if the patient is actually pregnant. At thesame time, pseudopod formation will not occur if the patient is notpregnant regardless of the fact of menstruation.

One should be especially careful in performing the test on sensitiveskins, as sometimes some skins will react to all proteins. This reactionmay be checked by making an extract of amnionic tissue which containsthe same amount of protein as in the original antigen, but is notcapable When individuals react to this latter extract they should not betested by the present method as their skins will probably react to anyprotein.

The finished antigen for the pregnancy test and the amnionic extract maybe heated to a temperature of about eighty degrees centigrade for aboutten minutes to destroy the hormones present.

The invention and discovery herein set forth designate to a high degreeof certainty whether or not a female is pregnant. The particular antigenherein described is made from the foetal layer only of the placenta andparticularly from the foetallayer of a placentafrom a born female. Theplacenta is obtained from the mother preferably not later than one hourafter birth, unless the same is frozen to prevent disintegration of thetissue cells, and while the exact method herein described is preferable,it is to be understood that various changes to a certain degree may bemade'without departing from the spirit of the invention or sacrificingany of its advantages, and the right is hereby reserved to make all suchchanges as fairly fall within the scope of the following claims.

The invention is hereby claimed as follows:

1. An antigen specific to the determination of pregnancy by intradermalinjection comprising a neutralized, inorganic alkaline hydroxide extractof the foetal-layer of placental tissue free from any portion of thematernal layer.

2. An antigen specific to the determination of pregnancy by intradermalinjection comprising a neutralized, inorganic alkaline hydroxide extractof the foetal layer of placental tissue free from any portion of thematernal layer, said antigen having a pH of substantially 6.9.

3. An antigen specific to the determination of pregnancy by intradermalinjection comprising a neutralized sodium hydroxide extract of thefoetal layer of mammalian placental tissue free from any portion of thematernal layer.

4. An antigen specific to the determination of pregnancy by intradermalinjection comprising a neutralized sodium hydroxide extract of thefoetal layer of human placental tissue free from any portion of thematernal layer, said antigen having a pH of substantially 6.9.

5. Theprocess of making an antigen for intradermal use to determine ifpregnancy exists which consists in extracting the foetal layer ofplacental tissue free from any portion of the maternal layer with aninorganic alkaline hydroxide, separating the extract, and thenneutralizing the extract.

6. The process of making an antigen for intradermal use to determine ifpregnancy exists which consists in obtaining an inorganic alkalineextract of the foetal layer of placental tissue free from any portion ofthe material layer, and then adding an acid and buffer solution toreduce the extract to a pH of approximately 6.9.

7. The process of making an antigen for intradermal use todetermine ifpregnancy exists comprising extracting the foetal layer of placentaltissue free from any portion of the material layer with sodiumhydroxide, separating the extract, and then neutralizing the extractwith potassium phosphate and hydrochloric acid.

8. The process of making an antigen for intradermal use to determine ifpregnancy exists comprising extracting the foetal layer of humanplacental tissue free from any portion of the maternal layer withone-tenth normal sodium hydroxide (NaOH), and then adding an acid andbufier solution of potassium phosphate (KH2PO4) and hydrochloric acid(HCl) to reduce the extract to a pH of substantially 6.9.

9. An antigen to determine if pregnancy exists comprising an extract ofthe foetal layer of mammalian placental tissue free fromany por-, tionof the maternal layer, said extract being adapted for intradermalinjection,-which extract contains a specific foetal embryonic proteinhomologous to the specific foetal embryonic protein of a pregnantmammalian and which produces a skin reaction by pseudopod formation whenthe said antigen is injected intradermally in a pregnant femalemammalian.

10. An antigen to determine if pregnancy exists comprising an extract ofthe foetal layer of mammalian placental tissue free from any portion ofthe maternal layer, said extract being adapted for intradermalinjection, which extract contains a specific foetal embryonic proteinhomologous to the specific foetal embryonic protein of a pregnantmammalian and which produces a skin reaction by pseudopod formation whenthe said antigen is injected intradermally in a-pregnant femalemammalian, said placentabeing a placenta from a born female.

BENJAMIN GRUSIHN.

